High performance liquid chromatography (HPLC) is, for many scientists, an essential piece of apparatus for the separation, identification, purification and quantification of various compounds. Users of HPLC work in a variety of fields including biomedical research, and the cosmetics, energy, food and environmental industries.
As outlined below, the origins of HPLC date back to the invention of chromatography in the early 20th century, through the introduction of partition and paper chromatography in the 1940s, to the introduction of liquid chromatography in the early 1960s. Shortly thereafter, the need for better resolution and high-speed analyses of nonvolatile samples led to the development of HPLC.
The Russian botanist Mikhail Tsvet is considered to have ‘invented’ the chromatographic technique when he reported separations of different plant pigments into a series of colored bands on a packed column. He called this technique ‘chromatography’.
Martin and Synge developed the theory of partition chromatography and used mathematics to describe the separation process resulting from the use of a liquid-coated solid phase and a moving liquid phase.
The technique of paper chromatography was developed by Consden, Gordon and Martin. This technique was originally used for the identification of amino acids.
J.C. Moore of the Dow Chemical Company was the first to investigate the technique of gel permeation chromatography.
The first commercial HPLC was manufactured by Waters Corporation, and was known as the ALC100 HPLC.
Dionex Corp. launched the first commercial ion-exchange chromatography system for the separation of ions and polar molecules based on their charge. The first Dionex ion-exchange columns benefited from enhanced ion detection capabilities through the use of revolutionary suppression technology that reduced background conductivity.
Cecil Tarbet (the founder of Cecil Instruments) introduced the world’s first commercially available variable wavelength monitor for HPLC.
By the late 1970s, new methods in HPLC, including reversed-phase liquid chromatography, allowed for improved separation between very similar compounds.
Agilent Technologies, Inc. introduced a new diode array detector, which provided a rapid optical method for chemical analysis.
HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far beyond previous techniques, while computers and automation provided convenience. Improvements in reproducibility were made as techniques such as micro-columns, affinity columns, and fast HPLC emerged.
ESA Biosciences, Inc. filed a patent for a new type of electrochemical detector known as the Coulochem.
Pharmacia (now GE Healthcare) developed the system of fast protein liquid chromatography (FPLC), a powerful chromatographic method that relies on pressures lower than those used in HPLC, making the technique more suitable for the separation of sensitive proteins.
HPLC gradually developed over the years, more by evolution than revolution. Incremental improvements combined to generate an extremely powerful tool capable of high precision and reproducibility. Some of the developments of the last 15 years are described below.
Waters introduced the Alliance ® HPLC system. Targeted mainly at pharmaceutical scientists concerned with the quality of their test results, the Alliance system was positioned as a product that raised the bar of performance by which HPLC would be measured. Alliance was subsequently named as “one of the most successful products in the history of analytical instruments” and one which “has been an important influence in fundamentally transforming the industry.”
Waters introduced the revolutionary XTerra® Column brand for drug development applications. XTerra Columns offered a new standard for high performance by giving pharmaceutical scientists greater speed, definable peak shapes and a usable pH range.
JASCO Corporation introduced the first ultra-high pressure HPLC pump, installed at Imperial College, London.
Waters unveiled a new category of LC technology known as Ultra Performance LC (UPLC) that would take the science of separation to a new level. This liquid chromatography system was the first of its kind, and designed to provide chromatographic run times up to ten times shorter than those of the fastest existing HPLC systems, with up to two times better peak capacity or resolution, and three times better routine sensitivity. For laboratories, these performance characteristics translated into more and higher-quality information per unit of time as well as greater productivity. A particle size of around 1.7 μm as used in UPLC allows greater speed and peak capacity, but also requires the use of a higher pressure to help move the eluent through the column.
Whatman, Inc. launched a faster and easier way to remove particulates from samples being prepared for HPLC through its Mini-UniPrep™ Syringeless Filter family. Mini-UniPrep was able to decrease sample batch processing time by one third for enhanced lab productivity, reducing the need for sample prep consumables, such as syringes, sample cups, and transfer containers.
Pickering Laboratories launched its sensitive method for identifying hard-to-detect chemical compounds, significantly advancing the science of analyte detection. At the heart of this development was the pulse-free syringe pump.
ESA Biosciences, Inc. launched the first new HPLC detection technology in 20 years, known as the ‘Corona Charged Aerosol Detector.’ In this system, the HPLC column eluent is nebulized and the resulting droplets are evaporated at ambient temperature producing analyte particles. A second stream of gas is positively charged as it passes a high-voltage, platinum corona wire. The charged gas collides with and transfers charges to the opposing stream of analyte particles. A negatively charged, low-voltage ion trap removes high-mobility ions while analyte particles transfer their charge to a collector. The charge transferred to the collector is in direct proportion to analyte mass. This system offers performance benefits that refractive index, low wavelength, evaporative light scattering, and chemiluminescence nitrogen detector methods lack.
JASCO launched the X-LC Xtreme HPLC system for use with sub-2 μm particles.
Agilent introduced a liquid chromatography system that removed the seven most abundant proteins in human plasma, unmasking previously undetectable proteins that are potential biological markers of drug toxicity or disease.
IDEX Health & Science launched a line of Ultra High Performance (UHP) fittings and connectors that increased the ability of a separation system to handle the demands of modern techniques. Used in applications requiring greater efficiency, speed and resolution, these UHP fittings and connectors effectively handled the stresses of higher temperatures and greater column pressures.
Agilent introduced the 1290 Infinity Liquid Chromatography System, designed to deliver significantly greater power, speed and sensitivity for enhanced performance in the high-end UHPLC market. Later the same year, Agilent also introduced the 1290 Infinity LC System sample injection system, offering superior performance in speed, ultra-low carryover and robustness for customers requiring high throughput. This injector extended sample capacity to 24 cooled microwell plates or 648 cooled 2-mL vials, for high-throughput usage.
Phenomenex introduced a unique way of achieving UHPLC performance from existing HPLC system hardware in certain applications with the Kinetex core-shell HPLC columns. Core-shell particles can be used with high mobile phase flow rates to further reduce analysis time without significant losses in separation efficiency, whereas the performance of fully porous particles begins to drop off sharply at high flow rates.
The Future of HPLC Systems
High performance liquid chromatography has been one of the defining separation techniques of the last 40 years, and its importance and range of uses will likely increase in the coming years.
Within biochemistry, fast and microbore columns will be developed for analytical and mass-scale preparative applications for biologicals and heterologously expressed gene products. Affinity and immunoaffinity techniques will be utilized more frequently for the production of biotechnological pharmaceuticals because of the need for ultrapurification in order to remove all unwanted material from the host cell. More accurate and higher capacity chiral separation columns will be needed by pharmaceutical companies in order to optimize efficiency in mass production of “active” enantiomeric compounds. HPLC will also have an important role in the monitoring of environmental pollutants.
Organic resins may become more widely used in the future. Multiple detectors may become standard per system, and computer-generated optimization of HPLC conditions will undoubtedly advance at the rate of computer technology. Use of robots may even eventually be used to handle and load hazardous items such as AIDS samples, viral/bacterial samples, radioisotopes, or environmental contaminants.
Whatever applications are developed, and no matter how HPLC continues to evolve, the technique looks certain to continue to be one of the most important laboratory separation techniques for analytical and preparative purposes in the future.
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